The present invention relates to polypeptide expression systems requiring cleavage of a precursor product, and to proteases for use in such systems. The present invention further relates to a novel polypeptide capable of reducing dichloroindophenol and oxidized glutathione, DNA encoding the novel polypeptide, vectors containing such DNA, hosts transformed with such vectors, and pharmaceutical compositions containing the polypeptide. In addition, the present invention also relates to monoclonal antibodies against this polypeptide, and a process for isolating and purifying the polypeptide using such an antibody.
The Potyviruses are a group of viruses each of which have a single-stranded, RNA genome of approximately 10,000 bases and which infects plants such as the family Solanaceae. The Potyvirus genome is characterized by possessing one extremely long open reading frame, or ORF, [Dougherty, W. G. and Hiebert, E. (1980), Virology 101:466-474.; Allison, R. et al. (1986), Virology 154: 9-20]. In order to express the individual proteins encoded within the ORF, the translated polyprotein is digested by two types of protease, both of which are also encoded within the ORF [Dougherty, W. G. and Carrington, J. C. (1988), Ann. Rev. Phytopath. 26: 23-143].
Tobacco etch virus (TEV) is a member of the Potyvirus family, and this virus produces nuclear inclusions which can be stained with trypan blue in the infected cell. The nuclear inclusions apparently consist of two kinds of protein, one of which has proven to be a viral protease, and which has been designated Nuclear Inclusion a, or NIa [J. Virol., 61:2540-2548 (1987)].
The Nuclear Inclusion a proteases of the Potyviruses recognize and cleave a peptide sequence which includes one of Gln-Gly, Gln-Ser and Gln-Ala, and it is believed that this sequence is hexameric and occurs at the C-terminal end of the relevant NIa within the polyprotein. Cleavage is between the two residues making up the dimers shown above.
The complete genomic sequences of TEV and tobacco vein mottling virus (TVMV), another member of the Potyvirus family, have been determined, and homology searching has allowed the location of the NIa""s of these viruses within their respective genomes [Virology, 154: 9-20 (1986); Nucleic Acid Res., 14:5417-5430 (1986)].
Clover Yellow Vein Virus, or CYVV, is also a Potyvirus. So far, only the gene occurring at the 3xe2x80x2 end of the CYVV genome, together with the coat protein it encodes, has been sequenced [Uyeda, I. et al. (1991), Intervirol. 32: 234-245]. The structure of NIa region of the genome has not previously been elucidated, nor has the corresponding NIa been isolated.
The production of exogenous proteins by expression systems can be straightforward, using techniques well known in the art. However, there are many polypeptides which cannot easily be expressed in an exogenous system. The problem may be that the polypeptide cannot be expressed in large amounts, and this cannot usually be corrected merely by placing a regulatory gene upstream. Alternatively, it may be that post-transcriptional events required to obtain the mature form do not take place, or take place incorrectly.
For example, many eukaryotic polypeptides are initially translated with an N-terminal methionine which is subsequently deleted to obtain the mature form. This processing cannot take place in prokaryotes, so that alternative means of obtaining expression have had to be found. One such technique involves fusing the desired exogenous protein with maltose-binding protein or glutathione S-transferase, for example, purifying the expressed fusion protein and then cleaving with a protease, such as Factor Xa, enterokinase, or thrombin. The main drawback of this cumbersome method is that it requires two purifiction steps, which results in a substantial loss of the end product.
U.S. Pat. No. 5,162,601 discloses the use of TEV protease in the manufacture of a polyprotein having linker sequences between each of the proteins it is desired to express, such as human tPA. However, this patent only discloses the cloning of a multigene encoding this polyprotein into a host. There is no disclosure of expression or purification of the proteolytically cleaved end product.
Oxygen for metabolic energy is generally provided in the form of oxidizing agents in the cellular environment. The activated form in which the oxygen is used is generally as a free radical, such as superoxide (O2xe2x88x92), peroxide (H2O2) or a hydroxy radical (OHxe2x88x92), all of which are reduced to form water (H2O) after use. Oxygen gas, itself, is highly oxidizing, but the term xe2x80x9cactivated oxygenxe2x80x9d, as used herein, relates to oxygen and oxygen-containing molecules which have greater oxidizing potential than atmospheric oxygen. The most potent form of activated oxygen is the free radical, which is a molecule or atom having one or more unpaired electrons.
Free radicals are typically unstable and, if not properly controlled, can denature lipids, proteins and nucleic acids. Consequently, although activated oxygen is essential to life, it is also a potential health hazard, and must be very closely controlled. Even in vanishingly small amounts, activated oxygen can cause disorders, due to high reactivity. As a result, living organisms are unable to survive unless they are equipped with a defence mechanism against activated oxygen.
In the cellular environment, the locations, amounts and times of generation of activated oxygen must be carefully balanced against the cell""s ability to neutralize the associated danger. This ability is typically provided by a defence mechanism that uses its own antioxidants or antioxidation enzymes. In the context of the present invention, an xe2x80x9cantioxidantxe2x80x9d is the generic name for a naturally occurring substance which is able to prevent or inhibit the auto-oxidation of lipids, for example. The term xe2x80x9cantioxidation enzymexe2x80x9d is used generically for an enzyme which catalyzes a reaction which eliminates activated oxygen, the term xe2x80x9cantioxidative actionxe2x80x9d being construed accordingly.
Excessive amounts of activated oxygen are produced in a number of abnormal circumstances, such as when a person is stressed, is taking drugs, smokes, undergoes surgery, has an organ transplant or if he suffers ischemia through a cerebral or myocardial infarction. These large amounts are more than the control systems of the body can eliminate, so that the excess of activated oxygen can cause further damage to the body, seriously impairing normal cells. The resulting, so-called oxidative stress is responsible for a great many disease conditions.
To take arteriosclerosis as an example, the occurrence of low specific gravity lipoproteins which have been oxidized by activated oxygen is considered to be one of the causes of the disease [Steinberg, D. (1983,) Arteriosclerosis 3, 283-301]. Oxidative stress is also considered to be intimately involved with cause and effect in the mechanisms associated with the occurrence, metabolic abnormalities and vascular complications of diabetes [Kondo, M. ed., xe2x80x9cApproaches from Modern Medicine (4) Free Radicalsxe2x80x9d, Medical View Pub., pp. 138-146].
Activated oxygen is also implicated in other pathological states and conditions, such as; ischemic disorders (reperfusion disorders, ischemic heart disease, cerebroischemia, ischemic enteritis and the like), edema, vascular hyperpermeability, inflammation, gastric mucosa disorders, acute pancreatitis, Crohn""s disease, ulcerative colitis, liver disorders, Paraquat""s disease, pulmonary emphysema, chemocarcinogenesis, carcinogenic metastasis, adult respiratory distress syndrome, disseminated intravascular coagulation (DIC), cataracts, premature retinopathy, auto-immune diseases, porphyremia, hemolytic diseases, Mediterranean anemia, Parkinson""s disease, Alzheimer""s disease, epilepsy, ultraviolet radiation disorders, radioactive disorders, frostbite and burns.
Several defence mechanisms exist both inside and outside cells for the sole purpose of eliminating activated oxygen generated physiologically.
Intracellularly, antioxidants and antioxidative enzymes, such as those given below, are known to process and eliminate activated oxygen. For example, catalase is present in peroxisomes, and this enzyme reduces and removes hydrogen peroxide. Glutathione peroxidase occurs in the cytoplasm and the mitochondria, and this enzyme reduces and detoxifies hydrogen peroxide and lipid peroxides in the presence of reduced glutathione. Transferrin, ferritin and lactoferrin, for example, inhibit the generation of activated oxygen by stabilizing iron ions, while ceruloplasmin performs a similar function in connection with copper ions. In addition, superoxide dismutase, which is present in the cytoplasm and mitochondria, catalyzes the reduction of superoxides to form hydrogen peroxide, the hydrogen peroxide then being eliminated by catalase, for example. In addition, vitamins C and E, reduced glutathione and other low molecular weight compounds are also capable of reducing and eliminating activated oxygen.
On the other hand, such agents as extracellular superoxide dismutase, extracellular glutathione peroxidase and reduced glutathione exist in the extracellular environment, and these have similar modes of action to their intracellular counterparts listed above. However, compared to the intracellular situation, there are fewer types of extracellular antioxidants and antioxidative enzymes, and there is only a small number that exhibit extracellular antioxidative action.
Reduced glutathione has an important function in maintaining the reduced state both inside and outside cells, and it is represented by the formula below. Glutathione was first discovered in yeast by de-Rey-Pailhade in 1888, and it was later named glutathione following its isolation as a compound by Hopkins in 1921. 
Glutathione is composed of three amino acids:xe2x80x94glutamic acid, cysteine and glycine. The thiol groups of two glutathione molecules can be oxidized to form a disulfide bond in the presence of activated oxygen, thereby reducing the activated oxygen.
Glutathione is mainly produced in the liver, and circulates within the body via the plasma. In the normal body, glutathione exists nearly entirely in the reduced form. When levels of the oxidized form increase, then the reduced form is regenerated by the action of glutathione reductase in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). Thus, reduced glutathione protects the cell membrane from oxidative disorders and functions by the reducing activated oxygen and free radicals. As a result of having this antioxidative property, reduced glutathione also protects against the effects of radioactivity and is useful as a therapeutic drug for cataracts. It has also recently been reported that systemic levels of reduced glutathione are reduced in AIDS patients, which tends to indicate that the role of reduced glutathione in the body is extremely important. However, in abnormal conditions, the amount of activated oxygen can be so great that virtually all glutathione is in the oxidized state, so that activated oxygen is not removed as fast as possible.
Human thioredoxin is a second example of a substance which exerts various physiological actions by means of its reducing activity, both inside and outside cells. Human thioredoxin (also known as Adult T Cell Leukemia Derived Factor, ADF) was cloned as a factor which was capable of inducing interleukin 2 receptors (IL-2R) in adult T cell leukemia cell lines. It is a thiol-dependent reductase with two cysteine residues at its active site, and it is capable of reducing activated oxygen and free radicals.
In addition to inducing IL-2 receptors, human thioredoxin also: promotes cell growth in B cell strain 3B6 which is infected with Epstein-Barr virus (EBV); protects against tumor necrosis factor (TNF) derived from monocyte-origin cell line U937; and protects against vascular endothelial cell impairment by neutrophils. Further, in the intracellular environment, human thioredoxin acts on the transcription factors NFkB, JUN and FOS through its reducing activity, thereby to promote DNA bonding activity and enabling it to function to increase transcriptional activity. Human thioredoxin is currently being developed as a protective agent for radioactivity disorders, as well as for a therapeutic drug for reperfusion disorders, rheumatoid arthritis and inflammations, all of which disorders can be protected against by its ability to protect against cellular impairment via its reducing activity.
As has been described above, it is physiologically extremely important to maintain both the intracellular and extracellular environments in a reduced state by elimination of activated oxygen and free radicals. There are believed to be many, as yet unknown, antioxidants and antioxidative enzymes, both inside and outside cells, that have a role to play in removing activated oxygen and free radicals. Accordingly, it would be extremely useful to find a reducing substance which was capable of regenerating, for example, reduced glutathione. Such a substance could help in abnormal bodily conditions, such as those described above.
It is a first object of the present invention to provide a novel protease, a nucleotide sequence encoding the protease, a vector containing a DNA sequence encoding the protease, and a host cell which has been transformed with said vector.
It is a second object of the present invention to provide DNA encoding a protein of interest and also encoding the novel protease upstream from said protein, a sequence between the two said sequences further encoding a peptide cleavable by said protease, all of said sequences being in the same open reading frame. It is also an object to provide a protein encoded by said DNA, as well as a vector containing said DNA and an expression system comprising said vector, said vector being able to replicate autonomously in an appropriate host cell, such as by comprising a nucleotide sequence required for autonomous replication.
In the alternative, it is a first object of the present invention to provide a nucleotide sequence encoding a novel polypeptide having reducing activity in vivo. It is also an object to provide such DNA which encodes a peptide which is capable of reducing dichloroindophenol and oxidized glutathione.
It is a further object of the present invention to provide a recombinant vector comprising the above-mentioned DNA, said vector being able to replicate autonomously in an appropriate host cell, such as by having a base sequence enabling autonomous replication.
Moreover, it is a yet further object of the present invention to provide a host cell microorganism which has been transformed with the above-mentioned recombinant vector. It is also an object to provide the above-mentioned peptide as an expression product from the transformed host cell, and to provide a monoclonal antibody against the peptide.
We have now identified and cloned the novel CYVV protease (NIa) and have surprisingly found that it is possible to use CYVV NIa as part of a fusion protein which can be expressed in such hosts as E. coli and which allows the production of large quantities of the fusion protein which can self-cleave to yield the desired exogenous protein. The CYVV NIa gene can be stably maintained and expressed in Escherichia coli, and the expressed NIa retains its activity as a specific protease, even when the protein forms part of a fusion protein.
We have also discovered DNA that codes for a novel polypeptide which is capable of reducing dichloroindophenol {also known as dichlorophenol-indophenol, 2,6-dichloroindophenol, or 2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one} and oxidized glutathione, said polypeptide being obtainable in large amounts by the use of appropriate genetic engineering techniques. This polypeptide is particularly useful in the therapy of conditions caused by, or related to, oxidative stress, or any disease caused by activated oxygen, such as arteriosclerosis, diabetes and ischemic disorders (including reperfusion disorders, ischemic cardiac diseases, cerebroischemia and ischemic enteritis).
Thus, in a first aspect of the first embodiment of the present invention, there is provided a polynucleotide sequence wherein said sequence comprises, in the 5xe2x80x2 to 3xe2x80x2 direction and in the same open reading frame:
a) a sequence encoding the clover yellow vein virus Nuclear Inclusion a protein, or a mutant or variant thereof having similar proteolytic specificity to that of clover yellow vein virus Nuclear Inclusion a protein;
b) a sequence encoding a peptide recognizable by and cleavable by said clover yellow vein virus Nuclear Inclusion a protein, or said mutant or variant thereof; and
c) at least one sequence encoding a polypeptide.
The present invention also provides a sequence encoding the clover yellow vein virus Nuclear Inclusion a protein, or a mutant or variant thereof having similar proteolytic specificity to that of clover yellow vein virus Nuclear Inclusion a protein.
The present invention further provides a vector, especially an expression vector, containing a sequence as defined above.
The present invention still further provides a host transformed with a vector as defined above, as well as an expression system comprising said host and said expression vector, and also a polypeptide produced by such an expression system.
In the alternative embodiment of the invention, there is provided, in a first aspect, a polynucleotide sequence encoding a polypeptide having the amino acid sequence of amino acid numbers 1 to 526 of sequence ID number 12, or which encodes a mutant or variant of said polypeptide, provided that the polypeptide encoded by the polynucleotide sequence is capable of reducing dichloroindophenol and oxidized glutathione.
There is also provided a vector, especially an expression vector, containing a sequence as defined above.
The present invention still further provides a host transformed with a vector as defined above, as well as an expression system comprising said host and said expression vector, and also a polypeptide produced by such an expression system.
The invention also provides the above polypeptide for use in therapy, as well as the use of such a polypeptide in the treatment and prophylaxis of conditions caused by, or related to, oxidative stress, or any disease caused by activated oxygen, and pharmaceutical compositions comprising the polypeptide.
There is yet further provided an antibody, especially a monoclonal antibody, and equivalents thereof, against the polypeptide, and the invention additionally provides a method of producing such an antibody and a method of purification of the polypeptide using the antibody.